1 Loading in Data sets + Library packages.

## 
## The downloaded binary packages are in
##  /var/folders/7n/x74qctp91rng390gx0z9hmd80000gn/T//RtmppF2EYo/downloaded_packages

2 Loading Dataset

options(future.globals.maxSize = 74 * 1024^3) # 55 GB
getOption("future.globals.maxSize") #59055800320
## [1] 79456894976
load(here("jk_code", "SO4_SUB_analysis.rds"))
Idents(SO4) <- "subclass2_MD"

SO4$sample <- factor(SO4$sample, levels = c("SO1", "SO4", "SO3", "SO2"))

VlnPlot(SO4,"S100g",split.by = "treatment",group.by = "sample")
## The default behaviour of split.by has changed.
## Separate violin plots are now plotted side-by-side.
## To restore the old behaviour of a single split violin,
## set split.plot = TRUE.
##       
## This message will be shown once per session.
## Warning: The `slot` argument of `FetchData()` is deprecated as of SeuratObject 5.0.0.
## ℹ Please use the `layer` argument instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
## Warning: `PackageCheck()` was deprecated in SeuratObject 5.0.0.
## ℹ Please use `rlang::check_installed()` instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.

# Macula Densa Markers , Nos1, Avpr1a, Slc12a1, Ptgs2, and Slc9a2

VlnPlot(SO4, c("Nos1","Avpr1a","Slc12a1","Ptgs2","Slc9a2","Pappa2"),split.by = "treatment",group.by = "treatment")

DimPlot(SO4)

SO4 <- SO4[
  !grepl("^(mt-|Rp|Gm)|Rik$", rownames(SO4)),
  ,
  drop = FALSE
]
## Warning: The following arguments are not used: drop
DimPlot(SO4,group.by = "treatment",split.by = "treatment")

#compare each type 1 control to type 1 low_treatment

3 Markers of control vs low_salt up/down regulation

Upregulated = low salt, positive, activated by low_salt Downregulated = control, negative, supressed of low_salt

VlnPlot(SO4, features = "Pappa2", group.by = "treatment", split.by = "subclass_MD")

DimPlot(SO4, reduction = "umap", split.by = "treatment", label = TRUE)

Idents(SO4) <- "treatment"

type_markers <- FindMarkers(
  object = SO4,
  ident.1 = "low_salt",   
  ident.2 = "control",     
  group.by = "treatment",
  min.pct = 0.1,           
  logfc.threshold = 0.1,  
  only.pos = FALSE         
)
## Warning: The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
## ℹ Please use the `layer` argument instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
## For a (much!) faster implementation of the Wilcoxon Rank Sum Test,
## (default method for FindMarkers) please install the presto package
## --------------------------------------------
## install.packages('devtools')
## devtools::install_github('immunogenomics/presto')
## --------------------------------------------
## After installation of presto, Seurat will automatically use the more 
## efficient implementation (no further action necessary).
## This message will be shown once per session
 type_markers$gene <- rownames(type_markers)
 
type_markers <-type_markers %>% arrange(desc(avg_log2FC))

3.1 Pathway Upregulated = Low salt

df<- type_markers 


df2 <- df %>% filter(p_val_adj < 0.05)


DEG_list <- df2

markers1up <- DEG_list %>% rownames_to_column(var="SYMBOL")


ENTREZ_list <- bitr(
  geneID = rownames(DEG_list),
  fromType = "SYMBOL",
  toType = "ENTREZID",
  OrgDb = org.Mm.eg.db
)
## 'select()' returned 1:1 mapping between keys and columns
## Warning in bitr(geneID = rownames(DEG_list), fromType = "SYMBOL", toType =
## "ENTREZID", : 2.76% of input gene IDs are fail to map...
markers1up <-  ENTREZ_list %>% inner_join(markers1up, by = "SYMBOL")

markers1up <-  markers1up %>% dplyr::filter(p_val_adj < 0.05)
#head(markers, n = 50)

pos.markers1up <-  markers1up %>% dplyr::filter(avg_log2FC > 0.4) %>%  arrange(desc(abs(avg_log2FC))) 
#change around avg log value 

pos.ranks1up <- pos.markers1up$ENTREZID[abs(pos.markers1up$avg_log2FC) > 0]
#head(pos.ranks)

pos_go1up <- enrichGO(gene = pos.ranks1up,           #a vector of entrez gene id
                   OrgDb = "org.Mm.eg.db",    
                   ont = "BP",
                   readable = TRUE)              #whether mapping gene ID to gene Name

pos_go1up
## #
## # over-representation test
## #
## #...@organism     Mus musculus 
## #...@ontology     BP 
## #...@keytype      ENTREZID 
## #...@gene     chr [1:131] "16010" "239435" "23850" "19283" "66815" "19225" "70337" ...
## #...pvalues adjusted by 'BH' with cutoff <0.05 
## #...417 enriched terms found
## 'data.frame':    417 obs. of  12 variables:
##  $ ID            : chr  "GO:0010563" "GO:0045936" "GO:0042326" "GO:0031400" ...
##  $ Description   : chr  "negative regulation of phosphorus metabolic process" "negative regulation of phosphate metabolic process" "negative regulation of phosphorylation" "negative regulation of protein modification process" ...
##  $ GeneRatio     : chr  "16/125" "16/125" "14/125" "15/125" ...
##  $ BgRatio       : chr  "388/28928" "388/28928" "337/28928" "420/28928" ...
##  $ RichFactor    : num  0.0412 0.0412 0.0415 0.0357 0.0411 ...
##  $ FoldEnrichment: num  9.54 9.54 9.61 8.27 9.52 ...
##  $ zScore        : num  11.16 11.16 10.48 9.88 10.03 ...
##  $ pvalue        : num  1.33e-11 1.33e-11 2.48e-10 4.44e-10 1.26e-09 ...
##  $ p.adjust      : num  1.84e-08 1.84e-08 2.28e-07 3.06e-07 6.97e-07 ...
##  $ qvalue        : num  1.29e-08 1.29e-08 1.60e-07 2.15e-07 4.89e-07 ...
##  $ geneID        : chr  "Hspb1/Cep85/Dusp1/Cdkn1c/Bmp2/Rgs2/Aldob/Cdk5rap1/Spry1/Jun/Ier3/Sh3bp5/Irf1/Gstp1/Dynll1/Ppp1r15a" "Hspb1/Cep85/Dusp1/Cdkn1c/Bmp2/Rgs2/Aldob/Cdk5rap1/Spry1/Jun/Ier3/Sh3bp5/Irf1/Gstp1/Dynll1/Ppp1r15a" "Hspb1/Cep85/Dusp1/Cdkn1c/Bmp2/Rgs2/Cdk5rap1/Spry1/Jun/Sh3bp5/Irf1/Gstp1/Dynll1/Ppp1r15a" "Hspb1/Cep85/Dusp1/Cdkn1c/Bmp2/Rgs2/Cdk5rap1/Hspa1b/Spry1/Jun/Sh3bp5/Irf1/Gstp1/Bex2/Ppp1r15a" ...
##  $ Count         : int  16 16 14 15 13 11 10 10 5 10 ...
## #...Citation
## G Yu. Thirteen years of clusterProfiler. The Innovation. 2024, 5(6):100722
 chart1up <- dotplot(pos_go1up) +
    ggtitle("upregulated lowsalt") +
    theme_classic() + 
    theme(
        plot.title = element_text(hjust = 0.5),
        legend.position = "left",
        axis.text.y = element_text(hjust = 0, size = 10)) +
    scale_y_discrete(position = "right", 
                     labels = function(x) str_wrap(x, width = 25))  # Wrap y-axis labels to 2 lines
## Scale for y is already present.
## Adding another scale for y, which will replace the existing scale.
chart1up

### Upregulation Gene View

pathway_type1up <- strsplit(pos_go1up$geneID, "/")

names(pathway_type1up) <- pos_go1up$Description 

pathway_type1up
## $`negative regulation of phosphorus metabolic process`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Aldob"    "Cdk5rap1" "Spry1"    "Jun"      "Ier3"     "Sh3bp5"  
## [13] "Irf1"     "Gstp1"    "Dynll1"   "Ppp1r15a"
## 
## $`negative regulation of phosphate metabolic process`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Aldob"    "Cdk5rap1" "Spry1"    "Jun"      "Ier3"     "Sh3bp5"  
## [13] "Irf1"     "Gstp1"    "Dynll1"   "Ppp1r15a"
## 
## $`negative regulation of phosphorylation`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Jun"      "Sh3bp5"   "Irf1"     "Gstp1"   
## [13] "Dynll1"   "Ppp1r15a"
## 
## $`negative regulation of protein modification process`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Hspa1b"   "Spry1"    "Jun"      "Sh3bp5"   "Irf1"    
## [13] "Gstp1"    "Bex2"     "Ppp1r15a"
## 
## $`negative regulation of protein phosphorylation`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Jun"      "Sh3bp5"   "Irf1"     "Gstp1"   
## [13] "Ppp1r15a"
## 
## $`negative regulation of transferase activity`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Zfp36"    "Spry1"    "Sh3bp5"   "Gstp1"   
## 
## $`negative regulation of protein kinase activity`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Sh3bp5"   "Gstp1"   
## 
## $`negative regulation of kinase activity`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Sh3bp5"   "Gstp1"   
## 
## $`protein refolding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5" 
## 
## $`cellular response to transforming growth factor beta stimulus`
##  [1] "Fos"    "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Wnt10a" "Spry1"  "Jun"   
##  [9] "Hspa5"  "Cited1"
## 
## $`transforming growth factor beta receptor signaling pathway`
## [1] "Fos"    "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Jun"    "Hspa5" 
## [9] "Cited1"
## 
## $`response to transforming growth factor beta`
##  [1] "Fos"    "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Wnt10a" "Spry1"  "Jun"   
##  [9] "Hspa5"  "Cited1"
## 
## $`chaperone-mediated protein folding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Clu"   
## 
## $`transforming growth factor beta receptor superfamily signaling pathway`
##  [1] "Fos"    "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Hfe"    "Bmper" 
##  [9] "Jun"    "Hspa5"  "Cited1"
## 
## $`positive regulation of cell projection organization`
##  [1] "Ptprz1"  "Ache"    "Hspb1"   "Rgs2"    "Scarf1"  "Hap1"    "Dynlt1f"
##  [8] "Lcn2"    "Dynll1"  "Hspa5"   "Robo2"   "Cx3cl1" 
## 
## $`regulation of epithelial cell differentiation`
## [1] "Cdkn1c" "Etv4"   "Bmp2"   "Sfn"    "Zfp36"  "F11r"   "Spry1"  "Cited1"
## 
## $`cell surface receptor protein serine/threonine kinase signaling pathway`
##  [1] "Fos"    "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Hfe"    "Bmper" 
##  [9] "Jun"    "Hspa5"  "Cited1"
## 
## $`negative regulation of cell adhesion`
##  [1] "Ptprz1" "Hspb1"  "Dusp1"  "Bcl6"   "Bmp2"   "Hfe"    "Irf1"   "Gstp1" 
##  [9] "Arg2"   "Acer2" 
## 
## $`negative regulation of MAP kinase activity`
## [1] "Dusp1" "Bmp2"  "Rgs2"  "Spry1" "Gstp1"
## 
## $`SMAD protein signal transduction`
## [1] "Fos"    "Bmp2"   "Hfe"    "Bmper"  "Jun"    "Cited1"
## 
## $`negative regulation of protein serine/threonine kinase activity`
## [1] "Dusp1"    "Bmp2"     "Rgs2"     "Cdk5rap1" "Spry1"    "Gstp1"   
## 
## $`negative regulation of neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Jun"    "Lcn2"   "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## 
## $`response to metal ion`
##  [1] "Ptgs2"   "Fos"     "Junb"    "Chp2"    "Aldob"   "Hspa8"   "Hfe"    
##  [8] "Jun"     "Trpc1"   "Pla2g4a"
## 
## $regeneration
## [1] "Mcub"   "Scarf1" "Hfe"    "Mymx"   "Jun"    "Cspg5"  "Gstp1"  "Cpq"   
## 
## $`prostaglandin transport`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Slco4a1"
## 
## $`ureteric bud development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`regulation of protein kinase activity`
##  [1] "Hspb1"    "Cep85"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Sfn"     
##  [7] "Rgs2"     "Cdk5rap1" "Spry1"    "Sh3bp5"   "Gstp1"   
## 
## $`positive regulation of neuron projection development`
## [1] "Ptprz1"  "Hspb1"   "Rgs2"    "Scarf1"  "Hap1"    "Dynlt1f" "Hspa5"  
## [8] "Cx3cl1" 
## 
## $`mesonephric epithelium development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`mesonephric tubule development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`chaperone cofactor-dependent protein refolding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5" 
## 
## $`mesonephros development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Hfe"    "Bmper"  "Hspa5" 
## 
## $`osteoclast differentiation`
## [1] "Fos"   "Junb"  "Bmp2"  "Klf10" "Anxa2" "Mafb" 
## 
## $`response to topologically incorrect protein`
## [1] "Hspb1"    "Hspa1a"   "Manf"     "Hspa5"    "Ppp1r15a" "Clu"     
## 
## $`cellular response to metal ion`
## [1] "Ptgs2" "Fos"   "Junb"  "Chp2"  "Hspa8" "Hfe"   "Jun"  
## 
## $`regulation of hemopoiesis`
##  [1] "Hspb1"  "Fos"    "Bcl6"   "Cd83"   "Hspa1b" "Zfp36"  "Klf10"  "Jun"   
##  [9] "Irf1"   "Mafb"  
## 
## $`negative regulation of type 2 immune response`
## [1] "Bcl6" "Irf1" "Arg2"
## 
## $`regulation of vesicle fusion`
## [1] "Syt5"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`'de novo' post-translational protein folding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5" 
## 
## $`regulation of transforming growth factor beta receptor signaling pathway`
## [1] "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Hspa5" 
## 
## $`'de novo' protein folding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5" 
## 
## $`response to calcium ion`
## [1] "Fos"     "Junb"    "Chp2"    "Jun"     "Trpc1"   "Pla2g4a"
## 
## $`negative regulation of cell-cell adhesion`
## [1] "Hspb1" "Bcl6"  "Bmp2"  "Hfe"   "Irf1"  "Gstp1" "Arg2" 
## 
## $`regulation of cellular response to transforming growth factor beta stimulus`
## [1] "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Hspa5" 
## 
## $`negative regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cav2"  "Bmp2"  "Bambi" "Spry1" "Bmper" "Hspa5"
## 
## $`response to heat`
## [1] "Ptgs2"  "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8" 
## 
## $`response to tumor necrosis factor`
## [1] "Ptgs2"  "Fos"    "Hspa1b" "Zfp36"  "Traf1"  "Irf1"   "Cx3cl1"
## 
## $`female pregnancy`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Pla2g4a" "Hsd11b2"
## 
## $`negative regulation of alpha-beta T cell activation`
## [1] "Bcl6" "Hfe"  "Irf1" "Arg2"
## 
## $`negative regulation of leukocyte cell-cell adhesion`
## [1] "Hspb1" "Bcl6"  "Hfe"   "Irf1"  "Gstp1" "Arg2" 
## 
## $`negative regulation of transforming growth factor beta receptor signaling pathway`
## [1] "Cav2"  "Bmp2"  "Bambi" "Spry1" "Hspa5"
## 
## $`response to starvation`
## [1] "Fos"   "Zfp36" "Hspa8" "Hfe"   "Klf10" "Jun"   "Hspa5"
## 
## $`prostaglandin secretion`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`regulation of inclusion body assembly`
## [1] "Hspa1b" "Hap1"   "Clu"   
## 
## $`regulation of neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Jun"    "Lcn2"   "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## 
## $`negative regulation of response to external stimulus`
## [1] "Dusp1"  "Sfn"    "Zfp36"  "Ier3"   "Gstp1"  "Arg2"   "Robo2"  "Anxa2" 
## [9] "Cx3cl1"
## 
## $`kidney epithelium development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`regulation of acute inflammatory response`
## [1] "Ptgs2"  "Ptges"  "C2cd4b" "Gstp1" 
## 
## $`plasma membrane organization`
## [1] "Cav2"  "Mymx"  "Rab3a" "Clu"   "Anxa2" "Mafb" 
## 
## $`negative regulation of apoptotic signaling pathway`
## [1] "Ptgs2"  "Hspb1"  "Hspa1b" "Ier3"   "Gstp1"  "Clu"    "Cx3cl1"
## 
## $`multi-organism reproductive process`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Pla2g4a" "Hsd11b2"
## 
## $`macrophage activation`
## [1] "Mcub"    "Jun"     "Pla2g4a" "Clu"     "Cx3cl1" 
## 
## $`protein folding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Clu"   
## 
## $`multi-multicellular organism process`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Pla2g4a" "Hsd11b2"
## 
## $`response to steroid hormone`
## [1] "Ptgs2"   "Fos"     "Junb"    "Zfp36"   "Hspa8"   "Jun"     "Gstp1"  
## [8] "Hsd11b2"
## 
## $`regulation of protein serine/threonine kinase activity`
## [1] "Dusp1"    "Bmp2"     "Sfn"      "Rgs2"     "Cdk5rap1" "Spry1"    "Gstp1"   
## 
## $`response to unfolded protein`
## [1] "Hspb1"    "Hspa1a"   "Manf"     "Hspa5"    "Ppp1r15a"
## 
## $`membrane fusion`
## [1] "Syt5"    "Cav2"    "Mymx"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`prostaglandin metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"  
## 
## $`prostanoid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"  
## 
## $`organ induction`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`acute inflammatory response`
## [1] "Ptgs2"  "Ptges"  "C2cd4b" "Hfe"    "Gstp1" 
## 
## $`regulation of lipid localization`
## [1] "Ptges"   "Lipg"    "Vstm2a"  "Pla2g4a" "Anxa2"   "Ttc39b" 
## 
## $`regulation of leukocyte differentiation`
## [1] "Hspb1" "Fos"   "Bcl6"  "Cd83"  "Klf10" "Jun"   "Irf1"  "Mafb" 
## 
## $`maternal process involved in female pregnancy`
## [1] "Ptgs2"   "Junb"    "Rgs2"    "Pla2g4a"
## 
## $`regulation of alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Hfe"  "Irf1" "Arg2"
## 
## $`positive regulation of lipid localization`
## [1] "Ptges"   "Lipg"    "Vstm2a"  "Pla2g4a" "Anxa2"  
## 
## $`kidney development`
## [1] "Cdkn1c" "Bmp2"   "Spry1"  "Hspa8"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`regulation of tumor necrosis factor production`
## [1] "Hspb1"  "Zfp36"  "Gstp1"  "Arg2"   "Clu"    "Cx3cl1"
## 
## $`regulation of tumor necrosis factor superfamily cytokine production`
## [1] "Hspb1"  "Zfp36"  "Gstp1"  "Arg2"   "Clu"    "Cx3cl1"
## 
## $`icosanoid transport`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Slco4a1"
## 
## $`positive regulation of vesicle fusion`
## [1] "Syt5"    "Pla2g4a" "Anxa2"  
## 
## $`negative regulation of muscle contraction`
## [1] "Ptgs2" "Rgs2"  "Arg2" 
## 
## $`negative regulation of leukocyte activation`
## [1] "Hspb1"  "Bcl6"   "Hfe"    "Irf1"   "Arg2"   "Cx3cl1"
## 
## $`positive regulation of fat cell differentiation`
## [1] "Ptgs2"  "Bmp2"   "Zfp36"  "Vstm2a"
## 
## $`tumor necrosis factor production`
## [1] "Hspb1"  "Zfp36"  "Gstp1"  "Arg2"   "Clu"    "Cx3cl1"
## 
## $`myeloid leukocyte differentiation`
## [1] "Fos"   "Junb"  "Bmp2"  "Klf10" "Jun"   "Anxa2" "Mafb" 
## 
## $`muscle cell proliferation`
## [1] "Ptgs2"  "Fos"    "Cav2"   "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1"
## 
## $`cellular response to tumor necrosis factor`
## [1] "Fos"    "Hspa1b" "Zfp36"  "Traf1"  "Irf1"   "Cx3cl1"
## 
## $`tumor necrosis factor superfamily cytokine production`
## [1] "Hspb1"  "Zfp36"  "Gstp1"  "Arg2"   "Clu"    "Cx3cl1"
## 
## $`renal system development`
## [1] "Cdkn1c" "Bmp2"   "Spry1"  "Hspa8"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
## 
## $`inclusion body assembly`
## [1] "Hspa1b" "Hap1"   "Clu"   
## 
## $`vesicle fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`regulation of membrane permeability`
## [1] "F11r"    "Hspa8"   "Dynlt1f" "Ier3"   
## 
## $`neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Jun"    "Lcn2"   "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## 
## $`phenol-containing compound metabolic process`
## [1] "Iyd"     "Trpc1"   "Slco4a1" "Cited1"  "Cpq"    
## 
## $`organelle membrane fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`positive regulation of secretion by cell`
## [1] "Ptges"   "Bmp2"    "Hfe"     "Trpc1"   "Pla2g4a" "Dynll1"  "Rab3a"  
## [8] "Anxa2"  
## 
## $`prostaglandin biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`regulation of animal organ formation`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`prostanoid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`cellular response to cadmium ion`
## [1] "Fos"   "Hspa8" "Jun"  
## 
## $`positive regulation of protein binding`
## [1] "Bmp2"  "Bambi" "Hfe"   "Anxa2"
## 
## $`regulation of inflammatory response`
## [1] "Ptgs2"  "Ptges"  "Bcl6"   "Zfp36"  "C2cd4b" "Ier3"   "Gstp1"  "Cx3cl1"
## 
## $decidualization
## [1] "Ptgs2"   "Junb"    "Pla2g4a"
## 
## $`regulation of supramolecular fiber organization`
## [1] "Hspa1a" "Hspa1b" "F11r"   "Limch1" "Hspa8"  "Capg"   "Clu"    "Cx3cl1"
## 
## $`negative regulation of T cell activation`
## [1] "Hspb1" "Bcl6"  "Hfe"   "Irf1"  "Arg2" 
## 
## $`positive regulation of leukocyte differentiation`
## [1] "Fos"   "Bcl6"  "Cd83"  "Klf10" "Jun"   "Irf1" 
## 
## $`positive regulation of hemopoiesis`
## [1] "Fos"   "Bcl6"  "Cd83"  "Klf10" "Jun"   "Irf1" 
## 
## $`regulation of type 2 immune response`
## [1] "Bcl6" "Irf1" "Arg2"
## 
## $`response to reactive oxygen species`
## [1] "Fos"   "Rhob"  "Hspa8" "Jun"   "Lcn2"  "Gstp1"
## 
## $`negative regulation of cell activation`
## [1] "Hspb1"  "Bcl6"   "Hfe"    "Irf1"   "Arg2"   "Cx3cl1"
## 
## $`negative regulation of cytokine production`
## [1] "Bcl6"   "Cd83"   "Zfp36"  "Hfe"    "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`regulation of response to endoplasmic reticulum stress`
## [1] "Manf"     "Hspa5"    "Ppp1r15a" "Clu"     
## 
## $dephosphorylation
## [1] "Ptprz1"   "Dusp1"    "Chp2"     "Bmp2"     "Ppm1j"    "Ppp1r15a"
## 
## $`cellular response to calcium ion`
## [1] "Fos"  "Junb" "Chp2" "Jun" 
## 
## $`response to toxic substance`
## [1] "Ptgs2" "Fos"   "Ptges" "Pon2"  "Lcn2"  "Gstp1"
## 
## $`regulation of myeloid cell differentiation`
## [1] "Fos"    "Hspa1b" "Zfp36"  "Klf10"  "Jun"    "Mafb"  
## 
## $`regulation of CD4-positive, alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Irf1" "Arg2"
## 
## $`regulation of lipid transport`
## [1] "Ptges"   "Lipg"    "Pla2g4a" "Anxa2"   "Ttc39b" 
## 
## $`response to oxidative stress`
## [1] "Ptgs2" "Hspb1" "Fos"   "Rhob"  "Hspa8" "Jun"   "Lcn2"  "Gstp1"
## 
## $`leukocyte cell-cell adhesion`
## [1] "Hspb1" "Bcl6"  "Cd83"  "F11r"  "Hfe"   "Irf1"  "Gstp1" "Arg2" 
## 
## $`skeletal muscle organ development`
## [1] "Mcub"  "Fos"   "Btg2"  "Cav2"  "Hspa8" "Mymx" 
## 
## $`regulation of cellular response to growth factor stimulus`
## [1] "Cdkn1c" "Cav2"   "Bmp2"   "Bambi"  "Spry1"  "Bmper"  "Hspa5" 
## 
## $`osteoblast differentiation`
## [1] "Ache"   "Junb"   "Bmp2"   "Tent5a" "Bambi"  "Cited1"
## 
## $`neuroinflammatory response`
## [1] "Ptgs2"  "Jun"    "Clu"    "Cx3cl1"
## 
## $`cellular response to reactive oxygen species`
## [1] "Fos"   "Rhob"  "Hspa8" "Jun"   "Lcn2" 
## 
## $`thyroid hormone metabolic process`
## [1] "Iyd"     "Slco4a1" "Cpq"    
## 
## $`response to interleukin-4`
## [1] "Mcm2"   "Hspa5"  "Cited1"
## 
## $`negative regulation of CD4-positive, alpha-beta T cell activation`
## [1] "Bcl6" "Irf1" "Arg2"
## 
## $`regulation of MAP kinase activity`
## [1] "Dusp1" "Bmp2"  "Rgs2"  "Spry1" "Gstp1"
## 
## $`response to glucocorticoid`
## [1] "Ptgs2"   "Fos"     "Zfp36"   "Gstp1"   "Hsd11b2"
## 
## $`regulation of metal ion transport`
## [1] "Mcub"     "Ptgs2"    "Hfe"      "Hap1"     "Trpc1"    "Lcn2"     "Cacna2d1"
## [8] "Cx3cl1"  
## 
## $`regulation of apoptotic signaling pathway`
## [1] "Ptgs2"  "Hspb1"  "Hspa1b" "Traf1"  "Ier3"   "Gstp1"  "Clu"    "Cx3cl1"
## 
## $`specification of animal organ identity`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`developmental induction`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`positive regulation of SMAD protein signal transduction`
## [1] "Bmp2"  "Hfe"   "Bmper"
## 
## $`positive regulation of animal organ morphogenesis`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`fatty acid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Lipg"    "Pla2g4a" "Gstp1"  
## 
## $`positive regulation of secretion`
## [1] "Ptges"   "Bmp2"    "Hfe"     "Trpc1"   "Pla2g4a" "Dynll1"  "Rab3a"  
## [8] "Anxa2"  
## 
## $`cellular response to chemical stress`
## [1] "Ptgs2" "Hspb1" "Fos"   "Rhob"  "Hspa8" "Jun"   "Lcn2" 
## 
## $`regulation of lens fiber cell differentiation`
## [1] "Cdkn1c" "Spry1" 
## 
## $`response to phorbol 13-acetate 12-myristate`
## [1] "Fos"  "Btg2"
## 
## $`cellular response to phorbol 13-acetate 12-myristate`
## [1] "Fos"  "Btg2"
## 
## $`keratinocyte differentiation`
## [1] "Ptgs2" "Etv4"  "Sfn"   "Zfp36" "Mafb" 
## 
## $`regulation of regulatory T cell differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`amide metabolic process`
## [1] "Degs2" "Aldob" "Hap1"  "Gstp1" "Arg2"  "Acer2" "Clu"   "Pmvk" 
## 
## $`cellular response to oxidative stress`
## [1] "Hspb1" "Fos"   "Rhob"  "Hspa8" "Jun"   "Lcn2" 
## 
## $`maternal placenta development`
## [1] "Ptgs2"   "Junb"    "Pla2g4a"
## 
## $`negative regulation of chemotaxis`
## [1] "Dusp1" "Gstp1" "Robo2"
## 
## $`positive regulation of release of sequestered calcium ion into cytosol`
## [1] "Hap1"   "Trpc1"  "Cx3cl1"
## 
## $`regulation of animal organ morphogenesis`
## [1] "Bmp2"   "Wnt10a" "Spry1"  "Robo2" 
## 
## $`positive regulation of phosphatase activity`
## [1] "Chp2" "Bmp2"
## 
## $`regulation of microtubule nucleation`
## [1] "Hspa1a" "Hspa1b"
## 
## $`regulation of fever generation`
## [1] "Ptgs2" "Ptges"
## 
## $`negative regulation of erythrocyte differentiation`
## [1] "Zfp36" "Mafb" 
## 
## $`positive regulation of unsaturated fatty acid biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`response to temperature stimulus`
## [1] "Ptgs2"  "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8" 
## 
## $`positive regulation of lipid transport`
## [1] "Ptges"   "Lipg"    "Pla2g4a" "Anxa2"  
## 
## $`negative regulation of lymphocyte activation`
## [1] "Hspb1" "Bcl6"  "Hfe"   "Irf1"  "Arg2" 
## 
## $`cellular response to epidermal growth factor stimulus`
## [1] "Fos"   "Zfp36" "Gstp1"
## 
## $`response to mechanical stimulus`
## [1] "Ptgs2" "Fos"   "Btg2"  "F11r"  "Jun"   "Irf1" 
## 
## $`regulation of endothelial cell migration`
## [1] "Ptgs2" "Hspb1" "Rhob"  "Bmper" "Lcn2" 
## 
## $`regulation of organic acid transport`
## [1] "Ptges"   "Rgs2"    "Pla2g4a" "Arg2"   
## 
## $`regulatory T cell differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`regulation of prostaglandin biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`cortisol metabolic process`
## [1] "Bmp2"    "Hsd11b2"
## 
## $`macrophage proliferation`
## [1] "Clu"    "Cx3cl1"
## 
## $`chaperone-mediated autophagy`
## [1] "Hspa8" "Clu"  
## 
## $`positive regulation of non-motile cilium assembly`
## [1] "Hap1"   "Dynll1"
## 
## $`negative regulation of MAPK cascade`
## [1] "Dusp1" "Bmp2"  "Rgs2"  "Spry1" "Gstp1"
## 
## $`alcohol metabolic process`
## [1] "Degs2"   "Bmp2"    "Pla2g4a" "Acer2"   "Hsd11b2" "Pmvk"    "Ttc39b" 
## 
## $`response to corticosteroid`
## [1] "Ptgs2"   "Fos"     "Zfp36"   "Gstp1"   "Hsd11b2"
## 
## $`regulation of neurogenesis`
## [1] "Ptprz1" "Ache"   "Btg2"   "Bmp2"   "Hap1"   "Robo2"  "Anxa2"  "Cx3cl1"
## 
## $`positive regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cdkn1c" "Bmp2"   "Hfe"    "Bmper" 
## 
## $`cell-substrate adhesion`
## [1] "Ptprz1" "Frem1"  "Bcl6"   "Limch1" "Cspg5"  "Acer2"  "Anxa2" 
## 
## $`response to BMP`
## [1] "Bmp2"   "Bambi"  "Hfe"    "Bmper"  "Vstm2a"
## 
## $`cellular response to BMP stimulus`
## [1] "Bmp2"   "Bambi"  "Hfe"    "Bmper"  "Vstm2a"
## 
## $`leukocyte proliferation`
## [1] "Junb"   "Bcl6"   "Irf1"   "Gstp1"  "Arg2"   "Clu"    "Cx3cl1"
## 
## $`response to endoplasmic reticulum stress`
## [1] "Manf"     "Jun"      "Hspa5"    "Nrbf2"    "Ppp1r15a" "Clu"     
## 
## $`carboxylic acid transport`
## [1] "Ptgs2"   "Ptges"   "Rgs2"    "Slc6a7"  "Pla2g4a" "Arg2"    "Slco4a1"
## 
## $`organelle fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`positive regulation of dephosphorylation`
## [1] "Chp2" "Bmp2"
## 
## $`positive regulation of nuclear-transcribed mRNA poly(A) tail shortening`
## [1] "Btg2"  "Zfp36"
## 
## $`microglial cell activation`
## [1] "Jun"    "Clu"    "Cx3cl1"
## 
## $`acute-phase response`
## [1] "Ptgs2" "Ptges" "Hfe"  
## 
## $`type 2 immune response`
## [1] "Bcl6" "Irf1" "Arg2"
## 
## $`regulation of erythrocyte differentiation`
## [1] "Hspa1b" "Zfp36"  "Mafb"  
## 
## $`organic acid transport`
## [1] "Ptgs2"   "Ptges"   "Rgs2"    "Slc6a7"  "Pla2g4a" "Arg2"    "Slco4a1"
## 
## $`mitotic nuclear division`
## [1] "Cep85"  "Dusp1"  "Cdkn1c" "Cav2"   "Hspa1a" "Hspa1b"
## 
## $`response to xenobiotic stimulus`
## [1] "Ptgs2"   "Fos"     "Hspa8"   "Jun"     "Lcn2"    "Gstp1"   "Acer2"  
## [8] "Hsd11b2"
## 
## $`response to epidermal growth factor`
## [1] "Fos"   "Zfp36" "Gstp1"
## 
## $`regulation of cyclin-dependent protein kinase activity`
## [1] "Cdkn1c"   "Sfn"      "Cdk5rap1"
## 
## $`positive regulation of endothelial cell migration`
## [1] "Ptgs2" "Hspb1" "Rhob"  "Lcn2" 
## 
## $`regulation of calcium ion transmembrane transport`
## [1] "Mcub"     "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"  
## 
## $`receptor-mediated endocytosis`
## [1] "Ache"  "Cav2"  "Hfe"   "Hap1"  "Clu"   "Anxa2"
## 
## $`unsaturated fatty acid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`icosanoid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`olefinic compound metabolic process`
## [1] "Ptgs2"   "Bmp2"    "Pla2g4a" "Gstp1"   "Hsd11b2"
## 
## $`fever generation`
## [1] "Ptgs2" "Ptges"
## 
## $`membrane raft assembly`
## [1] "Cav2"  "Anxa2"
## 
## $`regulation of iron ion transport`
## [1] "Hfe"  "Lcn2"
## 
## $`regulation of T-helper 2 cell differentiation`
## [1] "Bcl6" "Irf1"
## 
## $`response to parathyroid hormone`
## [1] "Fos"    "Cited1"
## 
## $`positive regulation of glial cell migration`
## [1] "Ptprz1" "Cx3cl1"
## 
## $`regulation of unsaturated fatty acid biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`regulation of leukocyte cell-cell adhesion`
## [1] "Hspb1" "Bcl6"  "Cd83"  "Hfe"   "Irf1"  "Gstp1" "Arg2" 
## 
## $`regulation of calcium ion transport`
## [1] "Mcub"     "Ptgs2"    "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"  
## 
## $`muscle organ development`
## [1] "Mcub"  "Fos"   "Btg2"  "Cav2"  "Bmp2"  "Hspa8" "Mymx" 
## 
## $`negative regulation of defense response`
## [1] "Sfn"    "Zfp36"  "Ier3"   "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`leukocyte activation involved in inflammatory response`
## [1] "Jun"    "Clu"    "Cx3cl1"
## 
## $`placenta development`
## [1] "Ptgs2"   "Junb"    "Cdkn1c"  "Pla2g4a" "Cited1" 
## 
## $`positive regulation of myeloid cell differentiation`
## [1] "Fos"    "Hspa1b" "Klf10"  "Jun"   
## 
## $`temperature homeostasis`
## [1] "Ptgs2" "Ache"  "Ptges" "Ksr2"  "Lcn2" 
## 
## $`regulation of smooth muscle cell proliferation`
## [1] "Ptgs2"  "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1"
## 
## $`icosanoid secretion`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`cellular response to heat`
## [1] "Ptgs2"  "Hspa1b" "Hspa8" 
## 
## $`response to cadmium ion`
## [1] "Fos"   "Hspa8" "Jun"  
## 
## $`positive regulation of prostaglandin secretion`
## [1] "Ptges"   "Pla2g4a"
## 
## $`regulation of odontogenesis of dentin-containing tooth`
## [1] "Bmp2"   "Wnt10a"
## 
## $`regulation of nuclear-transcribed mRNA poly(A) tail shortening`
## [1] "Btg2"  "Zfp36"
## 
## $`regulation of small molecule metabolic process`
## [1] "Igfbp4"  "Ptgs2"   "Bmp2"    "Aldob"   "Ier3"    "Pla2g4a" "Ttc39b" 
## 
## $`negative regulation of T cell differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`cellular response to starvation`
## [1] "Fos"   "Hfe"   "Klf10" "Jun"   "Hspa5"
## 
## $`regulation of intracellular transport`
## [1] "Ptgs2" "Chp2"  "Sfn"   "Hap1"  "Ier3"  "Anxa2"
## 
## $`regulation of epidermal cell differentiation`
## [1] "Etv4"  "Sfn"   "Zfp36"
## 
## $`positive regulation of blood pressure`
## [1] "F11r"    "Manf"    "Hsd11b2"
## 
## $`amino acid import across plasma membrane`
## [1] "Rgs2"   "Slc6a7" "Arg2"  
## 
## $`positive regulation of miRNA transcription`
## [1] "Fos"  "Bmp2" "Jun" 
## 
## $`alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Hfe"  "Irf1" "Arg2"
## 
## $`fatty acid transport`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Slco4a1"
## 
## $`regulation of non-motile cilium assembly`
## [1] "Hap1"   "Dynll1"
## 
## $`regulation of interleukin-1 production`
## [1] "Hspb1"  "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`negative regulation of leukocyte differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1"  "Mafb" 
## 
## $`negative regulation of cell cycle`
## [1] "Ptgs2"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rhob"     "Cdk5rap1" "Ier3"    
## 
## $`smooth muscle cell proliferation`
## [1] "Ptgs2"  "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1"
## 
## $`membrane assembly`
## [1] "Cav2"  "Anxa2" "Mafb" 
## 
## $`response to insulin`
## [1] "Ache"    "Fos"     "Cav2"    "Gstp1"   "Cited1"  "Hsd11b2"
## 
## $`regulation of fibroblast proliferation`
## [1] "Ptprz1" "Jun"    "Gstp1"  "Anxa2" 
## 
## $`axon regeneration`
## [1] "Scarf1" "Jun"    "Cspg5" 
## 
## $`p38MAPK cascade`
## [1] "Dusp1" "Bmp2"  "Zfp36"
## 
## $`glial cell activation`
## [1] "Jun"    "Clu"    "Cx3cl1"
## 
## $`heart field specification`
## [1] "Bmp2"  "Robo2"
## 
## $`regulation of translational fidelity`
## [1] "Cdk5rap1" "Lars2"   
## 
## $`regulation of heat generation`
## [1] "Ptgs2" "Ptges"
## 
## $`regulation of prostaglandin secretion`
## [1] "Ptges"   "Pla2g4a"
## 
## $`positive regulation of brown fat cell differentiation`
## [1] "Ptgs2"  "Vstm2a"
## 
## $`regulation of plasma membrane organization`
## [1] "Rab3a" "Anxa2"
## 
## $`lipid export from cell`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`regulation of proteasomal ubiquitin-dependent protein catabolic process`
## [1] "Hspa1a" "Hspa1b" "Hfe"    "Clu"   
## 
## $`interleukin-1 production`
## [1] "Hspb1"  "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`negative regulation of hemopoiesis`
## [1] "Hspb1" "Bcl6"  "Irf1"  "Mafb" 
## 
## $`negative regulation of cell-substrate adhesion`
## [1] "Ptprz1" "Bcl6"   "Acer2" 
## 
## $`regulation of macrophage activation`
## [1] "Mcub"    "Pla2g4a" "Cx3cl1" 
## 
## $`regulation of CD4-positive, alpha-beta T cell differentiation`
## [1] "Bcl6" "Cd83" "Irf1"
## 
## $`regulation of synaptic vesicle exocytosis`
## [1] "Syt5"  "Cspg5" "Rab3a"
## 
## $`positive regulation of DNA-binding transcription factor activity`
## [1] "Bmp2"   "Hspa1b" "Traf1"  "Bex2"   "Clu"   
## 
## $`regulation of protein ubiquitination`
## [1] "Arrdc4" "Hspa1b" "Nsmce3" "Bex2"   "Hspa5" 
## 
## $`lysosomal transport`
## [1] "Hspa1a" "Rhob"   "Hspa8"  "Clu"   
## 
## $`glycine transport`
## [1] "Rgs2"   "Slc6a7"
## 
## $`negative regulation of smooth muscle contraction`
## [1] "Ptgs2" "Rgs2" 
## 
## $`positive regulation of receptor-mediated endocytosis`
## [1] "Hfe"   "Clu"   "Anxa2"
## 
## $`brown fat cell differentiation`
## [1] "Ptgs2"  "Rgs2"   "Vstm2a"
## 
## $`positive regulation of miRNA metabolic process`
## [1] "Fos"  "Bmp2" "Jun" 
## 
## $`negative regulation of immune response`
## [1] "Bcl6" "Sfn"  "Hfe"  "Irf1" "Arg2"
## 
## $`skeletal muscle tissue development`
## [1] "Mcub"  "Fos"   "Btg2"  "Cav2"  "Hspa8"
## 
## $`positive regulation of epithelial cell differentiation`
## [1] "Etv4" "Bmp2" "Sfn" 
## 
## $`regulation of sterol transport`
## [1] "Lipg"   "Anxa2"  "Ttc39b"
## 
## $`regulation of cholesterol transport`
## [1] "Lipg"   "Anxa2"  "Ttc39b"
## 
## $`negative regulation of lymphocyte differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`regulation of epidermis development`
## [1] "Etv4"  "Sfn"   "Zfp36"
## 
## $`regulation of exocytosis`
## [1] "Syt5"  "Hap1"  "Cspg5" "Rab3a" "Anxa2"
## 
## $`morphogenesis of an endothelium`
## [1] "Rhob" "Lcn2"
## 
## $`endothelial tube morphogenesis`
## [1] "Rhob" "Lcn2"
## 
## $`negative regulation of amyloid-beta formation`
## [1] "Hap1" "Clu" 
## 
## $`monocarboxylic acid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Lipg"    "Pla2g4a" "Gstp1"  
## 
## $`CD4-positive, alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Irf1" "Arg2"
## 
## $`regulation of nucleocytoplasmic transport`
## [1] "Ptgs2" "Chp2"  "Sfn"   "Ier3" 
## 
## $`regulation of blood pressure`
## [1] "Ptgs2"   "F11r"    "Manf"    "Ier3"    "Hsd11b2"
## 
## $`small GTPase-mediated signal transduction`
## [1] "Bcl6"    "Rhob"    "F11r"    "Spry1"   "Dynlt1f" "Ksr2"    "Dock10" 
## 
## $`positive regulation of neurogenesis`
## [1] "Ptprz1" "Ache"   "Bmp2"   "Hap1"   "Robo2"  "Cx3cl1"
## 
## $`regulation of ERK1 and ERK2 cascade`
## [1] "Dusp1" "Bmp2"  "Spry1" "Bmper" "Jun"   "Gstp1"
## 
## $`positive regulation of transforming growth factor beta production`
## [1] "Ptgs2"  "Cx3cl1"
## 
## $`membrane biogenesis`
## [1] "Cav2"  "Anxa2" "Mafb" 
## 
## $`cell-cell adhesion via plasma-membrane adhesion molecules`
## [1] "Bmp2"   "Scarf1" "Pcdh1"  "Robo2"  "Cx3cl1"
## 
## $`bone mineralization`
## [1] "Ptgs2"  "Bmp2"   "Tent5a" "Klf10" 
## 
## $`regulation of receptor-mediated endocytosis`
## [1] "Hfe"   "Hap1"  "Clu"   "Anxa2"
## 
## $`regulation of cell-substrate adhesion`
## [1] "Ptprz1" "Bcl6"   "Limch1" "Cspg5"  "Acer2" 
## 
## $`regulation of chemotaxis`
## [1] "Hspb1"  "Dusp1"  "Gstp1"  "Robo2"  "Cx3cl1"
## 
## $`positive regulation of proteolysis`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Acer2"  "Clu"    "Anxa2" 
## 
## $`negative regulation of tumor necrosis factor production`
## [1] "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`regulation of monoatomic cation transmembrane transport`
## [1] "Mcub"     "Hap1"     "Trpc1"    "Lcn2"     "Cacna2d1" "Cx3cl1"  
## 
## $`regulation of amino acid import across plasma membrane`
## [1] "Rgs2" "Arg2"
## 
## $`negative regulation of mitochondrial membrane permeability`
## [1] "Dynlt1f" "Ier3"   
## 
## $`T-helper 2 cell differentiation`
## [1] "Bcl6" "Irf1"
## 
## $`regulation of receptor binding`
## [1] "Hfe"   "Anxa2"
## 
## $`negative regulation of amyloid precursor protein catabolic process`
## [1] "Hap1" "Clu" 
## 
## $`regulation of amino acid transmembrane transport`
## [1] "Rgs2" "Arg2"
## 
## $`regulation of CD8-positive, alpha-beta T cell activation`
## [1] "Hfe"  "Irf1"
## 
## $`endothelial cell migration`
## [1] "Ptgs2" "Hspb1" "Rhob"  "Bmper" "Lcn2" 
## 
## $`lipid transport`
## [1] "Ptgs2"   "Ptges"   "Lipg"    "Pla2g4a" "Slco4a1" "Anxa2"   "Ttc39b" 
## 
## $`protein dephosphorylation`
## [1] "Ptprz1"   "Dusp1"    "Ppm1j"    "Ppp1r15a"
## 
## $`neuron projection regeneration`
## [1] "Scarf1" "Jun"    "Cspg5" 
## 
## $`negative regulation of tumor necrosis factor superfamily cytokine production`
## [1] "Gstp1"  "Arg2"   "Cx3cl1"
## 
## $`cell-matrix adhesion`
## [1] "Frem1"  "Bcl6"   "Limch1" "Acer2"  "Anxa2" 
## 
## $`positive regulation by host of viral process`
## [1] "Cav2"  "Hspa8"
## 
## $`sphingoid metabolic process`
## [1] "Degs2" "Acer2"
## 
## $`regulation of protein-containing complex assembly`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Rab3a"  "Capg"   "Clu"   
## 
## $`regulation of small GTPase mediated signal transduction`
## [1] "Bcl6"    "F11r"    "Spry1"   "Dynlt1f" "Dock10" 
## 
## $`regulation of amide metabolic process`
## [1] "Aldob" "Hap1"  "Clu"  
## 
## $`regulation of SMAD protein signal transduction`
## [1] "Bmp2"  "Hfe"   "Bmper"
## 
## $`regulation of fat cell differentiation`
## [1] "Ptgs2"  "Bmp2"   "Zfp36"  "Vstm2a"
## 
## $`positive regulation of binding`
## [1] "Bmp2"  "Bambi" "Hfe"   "Anxa2"
## 
## $`skin development`
## [1] "Ptgs2"  "Etv4"   "Sfn"    "Wnt10a" "Zfp36"  "Mafb"  
## 
## $`animal organ formation`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`regulation of lipoprotein particle clearance`
## [1] "Lipg"  "Anxa2"
## 
## $`negative regulation of amide metabolic process`
## [1] "Hap1" "Clu" 
## 
## $`regulation of mitotic spindle assembly`
## [1] "Hspa1a" "Hspa1b"
## 
## $`tertiary alcohol metabolic process`
## [1] "Bmp2"    "Hsd11b2"
## 
## $`negative regulation of membrane permeability`
## [1] "Dynlt1f" "Ier3"   
## 
## $`regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains`
## [1] "Bcl6"    "Hspa8"   "Hfe"     "Pla2g4a" "Irf1"   
## 
## $`regulation of myeloid leukocyte differentiation`
## [1] "Fos"   "Klf10" "Jun"   "Mafb" 
## 
## $`fibroblast proliferation`
## [1] "Ptprz1" "Jun"    "Gstp1"  "Anxa2" 
## 
## $`calcium ion transport`
## [1] "Mcub"     "Ptgs2"    "Hap1"     "Tmem37"   "Trpc1"    "Cacna2d1" "Cx3cl1"  
## 
## $`regulation of nitric oxide biosynthetic process`
## [1] "Ptgs2"  "Dynll1" "Clu"   
## 
## $`cellular response to hydrogen peroxide`
## [1] "Rhob"  "Hspa8" "Lcn2" 
## 
## $`learning or memory`
## [1] "Ptprz1" "Ptgs2"  "Fos"    "Btg2"   "Jun"    "Lcn2"  
## 
## $`extrinsic apoptotic signaling pathway`
## [1] "Hspa1b" "Traf1"  "Lcn2"   "Gstp1"  "Cx3cl1"
## 
## $`positive regulation of myeloid leukocyte differentiation`
## [1] "Fos"   "Klf10" "Jun"  
## 
## $`positive regulation of nucleocytoplasmic transport`
## [1] "Ptgs2" "Chp2"  "Sfn"  
## 
## $`extrinsic apoptotic signaling pathway in absence of ligand`
## [1] "Hspa1b" "Lcn2"   "Cx3cl1"
## 
## $`regulation of miRNA transcription`
## [1] "Fos"  "Bmp2" "Jun" 
## 
## $`icosanoid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"  
## 
## $`positive regulation of intracellular transport`
## [1] "Ptgs2" "Chp2"  "Sfn"   "Anxa2"
## 
## $`organic anion transport`
## [1] "Ptgs2"   "Ptges"   "Rgs2"    "Slc6a7"  "Pla2g4a" "Arg2"    "Slco4a1"
## 
## $`positive regulation of icosanoid secretion`
## [1] "Ptges"   "Pla2g4a"
## 
## $`response to muscle stretch`
## [1] "Fos" "Jun"
## 
## $`regulation of odontogenesis`
## [1] "Bmp2"   "Wnt10a"
## 
## $`positive regulation of epidermal cell differentiation`
## [1] "Etv4" "Sfn" 
## 
## $`negative regulation of T-helper cell differentiation`
## [1] "Bcl6" "Irf1"
## 
## $`negative regulation of cyclin-dependent protein kinase activity`
## [1] "Cdkn1c"   "Cdk5rap1"
## 
## $`negative regulation of locomotion`
## [1] "Dusp1"  "Rhob"   "Limch1" "Gstp1"  "Robo2"  "Cx3cl1"
## 
## $`positive regulation of proteasomal ubiquitin-dependent protein catabolic process`
## [1] "Hspa1a" "Hspa1b" "Clu"   
## 
## $`positive regulation of fibroblast proliferation`
## [1] "Ptprz1" "Jun"    "Anxa2" 
## 
## $`L-alpha-amino acid transmembrane transport`
## [1] "Rgs2"   "Slc6a7" "Arg2"  
## 
## $`calcium ion transmembrane transport`
## [1] "Mcub"     "Hap1"     "Tmem37"   "Trpc1"    "Cacna2d1" "Cx3cl1"  
## 
## $`negative regulation of growth`
## [1] "Bcl6"   "Hspa1a" "Rgs2"   "Hspa1b" "Wwc2"  
## 
## $`organic hydroxy compound biosynthetic process`
## [1] "Bmp2"   "Trpc1"  "Cited1" "Acer2"  "Pmvk"  
## 
## $`ERK1 and ERK2 cascade`
## [1] "Dusp1" "Bmp2"  "Spry1" "Bmper" "Jun"   "Gstp1"
## 
## $`unsaturated fatty acid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"  
## 
## $`signal transduction in absence of ligand`
## [1] "Hspa1b" "Lcn2"   "Cx3cl1"
## 
## $`miRNA transcription`
## [1] "Fos"  "Bmp2" "Jun" 
## 
## $`regulation of nitric oxide metabolic process`
## [1] "Ptgs2"  "Dynll1" "Clu"   
## 
## $`regulation of protein modification by small protein conjugation or removal`
## [1] "Arrdc4" "Hspa1b" "Nsmce3" "Bex2"   "Hspa5" 
## 
## $`vascular process in circulatory system`
## [1] "Ptgs2"  "Rgs2"   "C2cd4b" "Manf"   "Cx3cl1"
## 
## $`regulation of phosphatase activity`
## [1] "Chp2" "Bmp2"
## 
## $`calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules`
## [1] "Bmp2"   "Cx3cl1"
## 
## $`melanin biosynthetic process`
## [1] "Trpc1"  "Cited1"
## 
## $`regulation of insulin-like growth factor receptor signaling pathway`
## [1] "Igfbp4" "Bmp2"  
## 
## $`nucleocytoplasmic transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Hspa8"  "Ier3"   "Cited1"
## 
## $`nuclear transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Hspa8"  "Ier3"   "Cited1"
## 
## $`regulation of alpha-beta T cell differentiation`
## [1] "Bcl6" "Cd83" "Irf1"
## 
## $`regulation of post-translational protein modification`
## [1] "Arrdc4" "Hspa1b" "Nsmce3" "Bex2"   "Hspa5" 
## 
## $`positive regulation of cation transmembrane transport`
## [1] "Hap1"   "Trpc1"  "Lcn2"   "Cx3cl1"
## 
## $`regulation of reactive oxygen species metabolic process`
## [1] "Ier3"  "Lcn2"  "Gstp1" "Arg2" 
## 
## $`epidermal cell differentiation`
## [1] "Ptgs2" "Etv4"  "Sfn"   "Zfp36" "Mafb" 
## 
## $`regulation of glial cell migration`
## [1] "Ptprz1" "Cx3cl1"
## 
## $`regulation of protein catabolic process`
## [1] "Hspa1a" "Hspa1b" "Hfe"    "Ier3"   "Clu"    "Anxa2" 
## 
## $`maintenance of location`
## [1] "Hap1"   "Vstm2a" "Trpc1"  "Hspa5"  "Cx3cl1" "Ttc39b"
## 
## $`regulation of osteoblast differentiation`
## [1] "Bmp2"   "Tent5a" "Bambi"  "Cited1"
## 
## $`regulation of adaptive immune response`
## [1] "Bcl6"    "Hspa8"   "Hfe"     "Pla2g4a" "Irf1"   
## 
## $`modulation by host of symbiont process`
## [1] "Cav2"  "Hspa8" "Jun"  
## 
## $`regulation of monoatomic ion transmembrane transport`
## [1] "Mcub"     "Hap1"     "Trpc1"    "Lcn2"     "Cacna2d1" "Cx3cl1"  
## 
## $`mRNA catabolic process`
## [1] "Btg2"   "Hspa1a" "Tent5a" "Hspa1b" "Zfp36" 
## 
## $`regulation of Rho protein signal transduction`
## [1] "Bcl6"   "F11r"   "Dock10"
## 
## $`regulation of release of sequestered calcium ion into cytosol`
## [1] "Hap1"   "Trpc1"  "Cx3cl1"
## 
## $`cell recognition`
## [1] "Ptprz1" "Fetub"  "Hspa1b" "Robo2" 
## 
## $`nuclear-transcribed mRNA poly(A) tail shortening`
## [1] "Btg2"  "Zfp36"
## 
## $`positive regulation of systemic arterial blood pressure`
## [1] "Manf"    "Hsd11b2"
## 
## $`melanin metabolic process`
## [1] "Trpc1"  "Cited1"
## 
## $`glucocorticoid metabolic process`
## [1] "Bmp2"    "Hsd11b2"
## 
## $`skeletal muscle cell proliferation`
## [1] "Fos"  "Cav2"
## 
## $`regulation of synaptic vesicle fusion to presynaptic active zone membrane`
## [1] "Syt5"  "Rab3a"
## 
## $`secondary metabolite biosynthetic process`
## [1] "Trpc1"  "Cited1"
## 
## $`regulation of brown fat cell differentiation`
## [1] "Ptgs2"  "Vstm2a"
## 
## $`regulation of synaptic vesicle membrane organization`
## [1] "Syt5"  "Rab3a"
## 
## $`response to ethanol`
## [1] "Fos"   "Rgs2"  "Hspa8" "Gstp1"
## 
## $`negative regulation of ERK1 and ERK2 cascade`
## [1] "Dusp1" "Spry1" "Gstp1"
## 
## $`vacuolar transport`
## [1] "Hspa1a" "Rhob"   "Hspa8"  "Clu"   
## 
## $`regulation of ubiquitin-dependent protein catabolic process`
## [1] "Hspa1a" "Hspa1b" "Hfe"    "Clu"   
## 
## $`erythrocyte differentiation`
## [1] "Bcl6"   "Hspa1b" "Zfp36"  "Mafb"  
## 
## $`negative regulation of protein-containing complex assembly`
## [1] "Hspa8" "Hspa5" "Capg"  "Clu"  
## 
## $`negative regulation of epithelial cell proliferation`
## [1] "Cdkn1c" "Etv4"   "Cav2"   "Sfn"   
## 
## $`hormone metabolic process`
## [1] "Iyd"     "Bmp2"    "Hfe"     "Slco4a1" "Cpq"    
## 
## $`endocardial cushion formation`
## [1] "Bmp2"  "Robo2"
## 
## $`protein targeting to lysosome`
## [1] "Hspa8" "Clu"  
## 
## $`membrane raft organization`
## [1] "Cav2"  "Anxa2"
## 
## $`peptidyl-tyrosine dephosphorylation`
## [1] "Ptprz1" "Dusp1" 
## 
## $`negative regulation of CD4-positive, alpha-beta T cell differentiation`
## [1] "Bcl6" "Irf1"
## 
## $`positive regulation of fatty acid biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`mitochondrion distribution`
## [1] "Hap1"   "Dynll1"
## 
## $`response to alcohol`
## [1] "Fos"   "Btg2"  "Rgs2"  "Hspa8" "Gstp1" "Pmvk" 
## 
## $`BMP signaling pathway`
## [1] "Bmp2"  "Bambi" "Hfe"   "Bmper"
## 
## $`fat cell differentiation`
## [1] "Ptgs2"  "Bmp2"   "Rgs2"   "Zfp36"  "Vstm2a"
## 
## $`cellular response to nutrient levels`
## [1] "Fos"   "Hfe"   "Klf10" "Jun"   "Hspa5"
## 
## $`heat generation`
## [1] "Ptgs2" "Ptges"
## 
## $`regulation of icosanoid secretion`
## [1] "Ptges"   "Pla2g4a"
## 
## $`positive regulation of epidermis development`
## [1] "Etv4" "Sfn" 
## 
## $`positive regulation of fatty acid transport`
## [1] "Ptges"   "Pla2g4a"
## 
## $`negative regulation of inflammatory response`
## [1] "Zfp36"  "Ier3"   "Gstp1"  "Cx3cl1"
## 
## $`nitric oxide biosynthetic process`
## [1] "Ptgs2"  "Dynll1" "Clu"   
## 
## $`myeloid leukocyte activation`
## [1] "Mcub"    "Jun"     "Pla2g4a" "Clu"     "Cx3cl1" 
## 
## $`regulation of extrinsic apoptotic signaling pathway`
## [1] "Hspa1b" "Traf1"  "Gstp1"  "Cx3cl1"
## 
## $`regulation of receptor recycling`
## [1] "Ache"  "Anxa2"
## 
## $`diol metabolic process`
## [1] "Degs2" "Acer2"
## 
## $`tissue regeneration`
## [1] "Mcub" "Mymx" "Cpq"

3.2 Downregulation Pathway

# Arrange and filter DEGs

df <- type_markers %>% arrange(desc(avg_log2FC))

df2 <- df %>% filter(p_val_adj < 0.05)

DEG_list <- df2

markers1down <- DEG_list %>% rownames_to_column(var="SYMBOL")

ENTREZ_list <- bitr(
  geneID = rownames(DEG_list),
  fromType = "SYMBOL",
  toType = "ENTREZID",
  OrgDb = org.Mm.eg.db
)
## 'select()' returned 1:1 mapping between keys and columns
## Warning in bitr(geneID = rownames(DEG_list), fromType = "SYMBOL", toType =
## "ENTREZID", : 2.76% of input gene IDs are fail to map...
markers1down <- ENTREZ_list %>% inner_join(markers1down, by = "SYMBOL")

markers1down <- markers1down %>% dplyr::filter(p_val_adj < 0.05)

pos.markers1down <- markers1down %>% dplyr::filter(avg_log2FC < 0) %>% arrange(desc(abs(avg_log2FC)))

pos.ranks1down <- pos.markers1down$ENTREZID[abs(pos.markers1down$avg_log2FC) > 0.4]

# GO enrichment for downregulated genes
pos_go1down <- enrichGO(
  gene = pos.ranks1down,
  OrgDb = org.Mm.eg.db,
  ont = "BP",
  readable = TRUE
)

pos_go1down
## #
## # over-representation test
## #
## #...@organism     Mus musculus 
## #...@ontology     BP 
## #...@keytype      ENTREZID 
## #...@gene     chr [1:41] "268709" "54156" "18703" "15395" "27528" "14077" "12709" ...
## #...pvalues adjusted by 'BH' with cutoff <0.05 
## #...32 enriched terms found
## 'data.frame':    32 obs. of  12 variables:
##  $ ID            : chr  "GO:0070374" "GO:0070372" "GO:0090287" "GO:0061077" ...
##  $ Description   : chr  "positive regulation of ERK1 and ERK2 cascade" "regulation of ERK1 and ERK2 cascade" "regulation of cellular response to growth factor stimulus" "chaperone-mediated protein folding" ...
##  $ GeneRatio     : chr  "5/38" "5/38" "5/38" "3/38" ...
##  $ BgRatio       : chr  "227/28928" "333/28928" "333/28928" "69/28928" ...
##  $ RichFactor    : num  0.022 0.015 0.015 0.0435 0.0138 ...
##  $ FoldEnrichment: num  16.8 11.4 11.4 33.1 10.5 ...
##  $ zScore        : num  8.65 6.94 6.94 9.68 6.61 ...
##  $ pvalue        : num  1.16e-05 7.21e-05 7.21e-05 1.03e-04 1.07e-04 ...
##  $ p.adjust      : num  0.013 0.024 0.024 0.024 0.024 ...
##  $ qvalue        : num  0.00892 0.01644 0.01644 0.01644 0.01644 ...
##  $ geneID        : chr  "Dusp15/Fgfbp3/Gas6/Notch2/Egf" "Dusp15/Fgfbp3/Gas6/Notch2/Egf" "Nrep/Fgf9/Fgfbp3/Sostdc1/Notch2" "Fkbp11/Umod/Pdcd5-ps" ...
##  $ Count         : int  5 5 5 3 5 5 2 2 4 2 ...
## #...Citation
## G Yu. Thirteen years of clusterProfiler. The Innovation. 2024, 5(6):100722
# Visualization
chart1down <- dotplot(pos_go1down) +
  ggtitle("type 1 downregulated control") +
  theme_classic() +
  theme(
    plot.title = element_text(hjust = 0.5),
    legend.position = "left",
    axis.text.y = element_text(hjust = 0, size = 10)
  ) +
  scale_y_discrete(
    position = "right",
    labels = function(x) str_wrap(x, width = 25)
  )
## Scale for y is already present.
## Adding another scale for y, which will replace the existing scale.
chart1down

3.2.1 Downreg Gene View

pathway_type1down <- strsplit(pos_go1down$geneID, "/")

names(pathway_type1down) <- pos_go1down$Description 

pathway_type1down
## $`positive regulation of ERK1 and ERK2 cascade`
## [1] "Dusp15" "Fgfbp3" "Gas6"   "Notch2" "Egf"   
## 
## $`regulation of ERK1 and ERK2 cascade`
## [1] "Dusp15" "Fgfbp3" "Gas6"   "Notch2" "Egf"   
## 
## $`regulation of cellular response to growth factor stimulus`
## [1] "Nrep"    "Fgf9"    "Fgfbp3"  "Sostdc1" "Notch2" 
## 
## $`chaperone-mediated protein folding`
## [1] "Fkbp11"   "Umod"     "Pdcd5-ps"
## 
## $`ERK1 and ERK2 cascade`
## [1] "Dusp15" "Fgfbp3" "Gas6"   "Notch2" "Egf"   
## 
## $`cell-substrate adhesion`
## [1] "Fam107a" "Egfl6"   "Plau"    "Plet1"   "Gas6"   
## 
## $`intracellular chloride ion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`intracellular monoatomic anion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`regulation of cell-substrate adhesion`
## [1] "Fam107a" "Egfl6"   "Plau"    "Plet1"  
## 
## $`phosphatidylcholine biosynthetic process`
## [1] "Fabp3" "Fabp5"
## 
## $`regulation of smooth muscle cell migration`
## [1] "Fgf9"  "Plau"  "Abhd2"
## 
## $`regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Nrep"    "Fgf9"    "Sostdc1" "Notch2" 
## 
## $`chloride ion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`smooth muscle cell migration`
## [1] "Fgf9"  "Plau"  "Abhd2"
## 
## $`plasminogen activation`
## [1] "Plau" "Pgk1"
## 
## $`monoatomic anion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`activation of protein kinase B activity`
## [1] "Gas6"   "Notch2"
## 
## $`regulation of cell-matrix adhesion`
## [1] "Fam107a" "Plau"    "Plet1"  
## 
## $`glomerular filtration`
## [1] "Umod" "Gas6"
## 
## $`muscle cell migration`
## [1] "Fgf9"  "Plau"  "Abhd2"
## 
## $`hepatocyte proliferation`
## [1] "Plau"   "Notch2"
## 
## $`epithelial cell proliferation involved in liver morphogenesis`
## [1] "Plau"   "Notch2"
## 
## $`embryonic limb morphogenesis`
## [1] "Hoxa10" "Fgf9"   "Notch2"
## 
## $`embryonic appendage morphogenesis`
## [1] "Hoxa10" "Fgf9"   "Notch2"
## 
## $`renal filtration`
## [1] "Umod" "Gas6"
## 
## $`liver morphogenesis`
## [1] "Plau"   "Notch2"
## 
## $`negative regulation of cell-matrix adhesion`
## [1] "Fam107a" "Plet1"  
## 
## $`regulation of smooth muscle cell differentiation`
## [1] "Fgf9"   "Notch2"
## 
## $`negative regulation of proteolysis`
## [1] "Ide"   "Plau"  "Timp3" "Gas6" 
## 
## $`gland morphogenesis`
## [1] "Plau"    "Sostdc1" "Notch2" 
## 
## $`transforming growth factor beta receptor superfamily signaling pathway`
## [1] "Nrep"    "Fgf9"    "Sostdc1" "Notch2" 
## 
## $`renal absorption`
## [1] "Umod" "Gas6"
# Start filtering process here
# Maybe use genes you discover from Upset Plot
# Maybe  try with subtypes fitler genes


gyarmati <- c("Fabp3", "Ccn1", "Foxq1", "Cxcl12", "Vash2", "Pamr1", "Vegfd", "Ccn3", "Bmp3", "Fgf9", "Spp1", "Wnt10a", "Sfrp1", "Tcf4", "Pappa2", "Unc5d", "Sema3c", "Robo2", "Slit2", "Egfl6", "Hgfac", "Pdgfc", "Megf9", "Frem1", "Thsd4", "Spock2", "Mmp14", "Adamtsl2", "Car8", "Irx1", "Irx2", "Hoxd11", "Hoxc4", "Etv1")

df3 <- type_markers %>%
  filter(gene %in% gyarmati) %>%
  arrange(desc(avg_log2FC))
  
df3 <- rownames(df3)

df <- AverageExpression(
  object = SO4,
  features = df3,
  group.by = 'treatment'
)$RNA
## As of Seurat v5, we recommend using AggregateExpression to perform pseudo-bulk analysis.
## Names of identity class contain underscores ('_'), replacing with dashes ('-')
## This message is displayed once per session.
df
## 24 x 2 sparse Matrix of class "dgCMatrix"
##               control     low-salt
## Pappa2   6.531402e+35 1.297082e+66
## Frem1    6.671330e-01 3.182392e+00
## Wnt10a   1.405144e+01 7.786316e+01
## Robo2    6.880488e+15 1.192981e+20
## Hoxc4    8.287261e-01 1.074161e+00
## Megf9    1.417514e+02 6.069250e+03
## Irx1     5.092308e+16 3.253313e+20
## Etv1     2.188832e+01 6.456954e+01
## Irx2     2.387772e+10 5.763812e+09
## Bmp3     2.217383e+07 1.102390e+09
## Sfrp1    8.518389e+87 3.659043e+88
## Vegfd    3.173728e+00 5.141249e+00
## Hgfac    1.091375e+00 1.701657e+00
## Vash2    3.906250e+00 3.524595e+01
## Thsd4    3.156320e+02 1.557459e+02
## Spock2   8.782243e+01 8.666974e-01
## Adamtsl2 2.425374e+00 9.739461e+00
## Mmp14    2.903324e+00 4.851085e+00
## Pamr1    7.977601e+05 6.994911e+07
## Hoxd11   2.683656e+00 4.938132e+00
## Cxcl12   1.781695e+00 1.482061e+00
## Fgf9     7.308532e+04 4.224723e+06
## Fabp3    8.046356e+06 1.633769e+06
## Egfl6    1.319648e+01 3.037382e+00
df <- as.data.frame(df)

df <- t(scale(t(df)))

# convert df to tidy format

# only use about 10-15 genes

df_tidy <- df %>%
  as.data.frame() %>%
  rownames_to_column(var = "Gene") %>%
  pivot_longer(cols = -Gene, names_to = "treatment", values_to = "Expression")
               
# Graph with geom_tile

df_tidy$treatment <- factor(df_tidy$treatment, levels = c("control","low-salt"))

f4 <- ggplot(df_tidy, aes(x = treatment, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +  # Adds thin border and spacing between tiles
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),         # Removes tick marks
    panel.grid = element_blank(),          # Removes grid lines
    
    legend.position = "bottom",            # Centers the legend below the plot
    legend.title = element_text(size = 14), # Increases legend title size
    legend.text = element_text(size = 12)   # Increases legend text size
  ) +
  labs(x = "Cell Type", y = "Gene", fill = "Relative Expression")
## Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
## ℹ Please use `linewidth` instead.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
f4

df3 <- type_markers %>%
  filter(gene %in% gyarmati) %>%
  arrange(desc(avg_log2FC))

df3 <- rownames(df3)

df <- AverageExpression(
  object = SO4,
  features = df3,
  group.by = 'sample'
)$RNA

df
## 24 x 4 sparse Matrix of class "dgCMatrix"
##                   SO1          SO4          SO3          SO2
## Pappa2   1.624997e+27 9.394787e+35 4.577074e+50 4.328080e+66
## Frem1    5.032261e-01 7.389903e-01 2.455748e+00 4.880402e+00
## Wnt10a   4.968846e+00 1.803328e+01 1.096020e+02 3.696514e+00
## Robo2    2.257493e+16 3.797770e+08 2.280856e+13 3.980717e+20
## Hoxc4    1.162726e+00 6.822996e-01 1.044906e+00 1.142524e+00
## Megf9    1.201802e+02 1.512083e+02 7.927695e+03 1.726471e+03
## Irx1     5.999075e+13 7.322160e+16 2.516864e+17 1.084971e+21
## Etv1     3.684967e+00 2.986872e+01 2.282637e+01 1.621142e+02
## Irx2     5.833102e+10 8.773306e+09 1.023481e+09 1.684093e+10
## Bmp3     5.780747e+07 6.551941e+06 3.438930e+08 2.874832e+09
## Sfrp1    2.794890e+88 3.417901e+78 5.224841e+88 1.153674e+84
## Vegfd    2.107406e+00 3.641206e+00 4.451030e+00 6.754140e+00
## Hgfac    1.714995e+00 8.179782e-01 1.470009e+00 2.242968e+00
## Vash2    2.142977e+00 4.679274e+00 7.986470e+00 9.894542e+01
## Thsd4    3.644712e+01 4.380274e+02 1.763989e+02 1.074844e+02
## Spock2   2.846748e-01 1.261992e+02 8.603720e-01 8.814787e-01
## Adamtsl2 2.292680e+00 2.483547e+00 8.617702e+00 1.236077e+01
## Mmp14    2.731674e+00 2.978576e+00 4.177641e+00 6.424778e+00
## Pamr1    2.401602e+06 9.463135e+04 5.502337e+07 1.048273e+08
## Hoxd11   4.309507e+00 1.970879e+00 4.240257e+00 6.568913e+00
## Cxcl12   4.084340e+00 7.722092e-01 9.820518e-01 2.650473e+00
## Fgf9     2.153550e+04 9.568490e+04 4.299888e+04 1.399650e+07
## Fabp3    1.983636e+07 2.877586e+06 2.183898e+06 3.482377e+05
## Egfl6    1.422648e+01 1.274492e+01 1.538748e+00 6.539362e+00
df <- as.data.frame(df)

df <- t(scale(t(df)))

# convert df to tidy format

df_tidy <- df %>%
  as.data.frame() %>%
  rownames_to_column(var = "Gene") %>%
  pivot_longer(cols = -Gene, names_to = "sample", values_to = "Expression")
               

# Graph with geom_tile

df_tidy$sample <- factor(df_tidy$sample, levels = c("SO1","SO4","SO2","SO3"))

f4 <- ggplot(df_tidy, aes(x = sample, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +  # Adds thin border and spacing between tiles
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),         # Removes tick marks
    panel.grid = element_blank(),          # Removes grid lines
    legend.position = "bottom",            # Centers the legend below the plot
    legend.title = element_text(size = 14), # Increases legend title size
    legend.text = element_text(size = 12)   # Increases legend text size
  ) +
  labs(x = "Sample", y = "Gyarmati Genes", fill = "Relative Expression")

f4

4 Grouping as Treatment groups

# 1. Create new sample groups

treatmentgroup <- df_tidy %>%
  mutate(
    sample = case_when(
      sample %in% c("SO1", "SO4") ~ "SO1_SO4",
      sample %in% c("SO2", "SO3") ~ "SO2_SO3"
    )
  ) %>%
  
  # 2. Sum (or use mean) of Expression within each group for each Gene
  group_by(Gene, sample) %>%
  summarize(Expression = mean(Expression), .groups = "drop")  # Use mean() if you prefer averaging

treatmentgroup$sample <- factor(treatmentgroup$sample, levels = c("SO1_SO4", "SO2_SO3"))
f4_grouped <- ggplot(treatmentgroup, aes(x = sample, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),
    panel.grid = element_blank(),
    legend.position = "bottom",
    legend.title = element_text(size = 14),
    legend.text = element_text(size = 12)
  ) +
  labs(x = "Control                                        Low Salt ", y = "Gyarmati Genes", fill = "Relative Expression")

f4_grouped

# HeatMap of each clusters top genes from pathway analysis

VlnPlot(SO4,split.by = "treatment","Wnt10a")